Background and Significance Nanoparticles (particles less than 100 nm) have generated increasing interest in the field of medicine as tools for disease detection, and drug delivery. The field of nanomedicine, while still in its infancy, holds tremendous promise as we begin to understand issues related to the benefits, toxicity and environmental impact of nanoscale materials. Colloidal gold is a neutral gold particle synthesized through the combination of gold chloride and sodium citrate. It has been used safely for decades as a therapeutic for patients with arthritis. The particle measures 5-100 nm in diameter and can be linked irreversibly to proteins, peptides, synthetic drugs and nucleotides. In addition to its properties as a nano-carrier, colloidal gold, being a high-Z element, may also increase the radiation dose delivered specifically to the target cells. Theoretically, the irradiation of high-Z elements at their K-edge absorption energy leads to emission of Auger electrons and photoelectrons, releasing a large amount of energy at their immediate vicinity. This secondary radiation will result in extra damage to tumor cells at the same dose of the applied external radiation. If systemic administration of tumor-targeted gold nanoparticles results in the preferential trafficking of the particles to tumor tissue, we will be able to utilize this property to enhance the efficacy of external beam irradiation by increasing the radiation dose deposited into the tumor while minimizing toxicity to normal tissues. Since the high amount of energy deposited by Auger electrons at short distances will break chemical binding of molecules attached to the gold nanoparticles, gold nanoparticles could be also used as delivery vehicles for radiation-triggered release of therapeutic agents. In addition, gold can be easily activated with thermal neutrons to emit 411 keV gamma rays, to provide a means for monitoring their biodistribution by SPECT. Therefore, in addition to a possible direct effect of Auger electrons on tumor cells, our nanoparticles activated for imaging of their distribution, containing tumor-specific targeting agents for selective delivery, and releasing the active drug within a target volume only at optimal time (defined by real-time imaging), may provide means for tumor-specific delivery of a variety of tumorocidal agents, including those whose application is currently limited due to their hydrophobicity and, thereby, complement current therapeutic strategies and, specifically, argument the efficacy of radiation therapy. This project is a joint effort encompassing a range of different disciplines which include: nuclear physics, analytical chemistry, cell surface receptor recognition, growth factor signaling pathways, targeted therapy, immunology pertaining to mAbs and affibody, protein chemistry, medicinal chemistry, radiochemistry, radiology, and molecular imaging. We have combined the strengths of the CCR Radiation Oncology Branch, Molecular Imaging and Nanobiology Programs, with NIH Imaging Probe Development Center, and NIST Analytical Chemistry Division to merge the project into an integrated approach for targeted drug delivery. As we move the project towards the clinical arena we will partner with CCR clinical radiation and medical oncologists for appropriate therapeutic strategies. Research Design Aim 1: Radiation transport calculations will be carried out using the ESG4 Monte Carlo simulation package or the treatment planning software developed within the ROB Radiation Physics Section. The changes of the radiation dose deposited in tissues as a function of the concentration of gold and the quality of the applied external irradiation will be calculated using standard methodology . Aim 2: This aim will be carried out in collaboration with Dr. Garry Griffith and his team at NIH Imaging Probe Development Center. Gold nanoparticles will be pegylated to prevent their aggregation and conjugated with HER2- or EGFR-specific affibody molecules. For imaging, near-infrared fluorescent molecules (e.g. AlexaFluor) will be attached, too. If the biodistribution studies give promising results, the nanoparticles will be loaded with therapeutic agents. Aim 3: Gold nanoparticles, conjugated with fluorescent dyes via different types of linkers, will be exposed to increasing doses of x-rays. After irradiation, the nanoparticles will be separated from solution containing molecules released form gold by x-ray-triggered Auger electrons. The florescence of these molecules will be measured and correlated with the exposure to x-rays. Aim 4: Response of tumor cells to combination of pegylated colloidal gold nanoparticles with various types of radiation will be characterized by the clonogenic survival assay. Human breast cancer cell lines will be incubated with or without gold nanoparticles, and exposed to different doses of radiation. The energy of applied x-rays will vary in the range from 5 keV to 60 MeV. The effects of radiation on cell survival will be quantified and evaluated. This series of experiments will help in the validation of the predicted doses from Aim 1 and will allow for the selection of the appropriate cell line, and radiation type and dose for in vivo studies. Aim 5: This work will be carried out in collaboration with Dr. R. Gregory Downing and his team at Analytical Chemistry Division of the National Institute of Standards and Technology. Using the thermal neutron port at NIST nuclear reactor gold nanoparticles will be irradiated with different fluencies of neutrons and their activation assess by measurement of the gamma rays emitted from the activated gold. The collateral damage to the molecules previously attached to the gold nanoparticles will be assessed by changes in physico-chemical characteristics of theses nanoparticles post-irradiation. Aim 6: Biodistribution of nanoparticles in tumor bearing mice will be studied ex vivo and in vivo using optical imaging and single-photon emission computer tomography (SPECT) for nanoparticles labeled with near-infrared fluorescent beacons and activated with neutrons, respectively. Aim 7: The possible synergistic effect of combining x-rays gold nanoparticles will be studied in vivo using tumor bearing mice. Groups of tumor-bearing animals will be treated with: 1) radiation only;2) gold only;and 3) combination radiation plus gold. Non-treated animals will be used as controls. Tumor size and survival will be used to assess the efficacy of each treatment. Aim 8: To assess the feasibility of gold nanoparticles for delivery of therapeutic agents and efficacy of the proposed radiation-triggered drug delivery system we will compare the therapeutic efficacy of the proposed radiation-triggered drug delivery system with standard therapeutic interventions in tumor bearing mice. Accomplishments: 1. Methods for quantification of gold concentration in the tissue using neutron activation have been developed and presented on 2009 Annual Meetings of the American Nuclear Society and American Chemical Society 2. Methods for production of pegylated nanoparticles have been established and tested 3. X-ray-triggered release of fluorescent molecules from gold nanoparticles was tested and a poster presentation of the preliminary results received Best American Chemical Society (ACS) poster at the ACS Division of Colloid and Surface on the 2009 Annual Meetings of ACS.